An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

1. General methods and material

1. Prepare the appropriate medium for the chosen mammalian cell culture and pre-warm it at 37 °C for 20 min before use.
2. Prepare the required material in advance, as described in the Table of Materials. Autoclave glassware, plasticware, and buffer stocks.
3. Prepare the buffers as described in Table 1. Adjust the pH of the stock solutions using concentrated HCl or NaOH before diluting the components to their final volumes.

2. Mammalian cell line preparation

1. Grow HEK 293T cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 µg/mL penicillin/streptomycin solution. Incubate them at 37 °C in a humidified incubator supplied with 5% CO₂. Routinely split the cells.
2. Before detaching, rinse the cells with enough phosphate-buffered saline (PBS) solution to cover the growing surface.
3. Remove the PBS and add an ultra-thin layer of trypsin-EDTA solution.
4. Incubate the cells at 37 °C in a humidified incubator provided with 5% CO₂ for 5 min, or until the cells are detached (they should look dispersed under microscopic observation).
5. Dilute the trypsin-EDTA solution tenfold by adding complete DMEM to inactivate it and count the cells.
6. Plate 1.5 x 10⁵ cells/mL in 6-well plates, considering two wells/condition to test.
7. Incubate the cells at 37 °C for 48 h in a humidified incubator with 5% CO₂.
   NOTE: Check the manufacturer instructions for the type of medium and supplement apt for the cell line. Also, the amount and the incubation time of trypsin-EDTA depend on the cell line. Some cell types grow faster/slower than what was reported in this protocol; thus, seeding concentration should be tested beforehand.

3. Total protein harvest

1. Remove medium from the wells where cells are growing.
2. Wash each well of the 6-well plates with 1 mL of PBS.
3. Discard the PBS.
4. Move the plates on ice and either proceed with step 3.5 or freeze the dry plates at -80 °C to facilitate lysis.
5. Add 200 µL of lysis buffer to each well.
6. Use a cell scraper to detach and break the cells.
7. Transfer the cell extract deriving from two wells into the same 1.5 mL tube.
8. Place the tube containing the protein extract on ice for 30 min.
9. Centrifuge the cell lysates at 17,000 x g for 15 min at 4 °C.
10. Transfer each supernatant into a pre-cooled tube.
    NOTE: A total of 10⁶-10⁷ cells for each PD condition are recommended. Cell lysis and protein harvest should be performed with ice-cold buffers. Protease inhibitors should be added to the lysis buffer to prevent protein degradation.

4. Protein concentration determination

1. Prepare Bradford reagent, as indicated by the producer, by diluting it fivefold in dH₂O.
2. Distribute 1 mL of reagent in a 1 cm cuvette, add 1 µL of the sample, and mix by inversion.
3. Incubate in the dark at room temperature for 5-10 min.
4. Read the absorbance at 595 nm.
5. Calculate the volume of protein extract corresponding to 1.5 mg of proteins and bring all the samples to a final volume of 600 µL using lysis buffer.
6. Keep the samples on ice until use.
   NOTE: Any other protein concentration determination method can be used, following buffer compatibility recommendations. In any case, the lysis buffer should be used as a blank. Many reagents are not compatible with dithiothreitol (DTT). It is recommended to add DTT or other reducing agents only after protein quantification (DTT to a final concentration of 1 mM).

5. Bead preparation

1. Mix the beads in their storage buffer by flicking the tube.
2. Calculate 100 µL of slurry medium/sample and place the volume into a magnetic rack.
3. Bead wash
   1. Remove the storage solution and wash the beads by adding 1 mL of lysis buffer/tube and invert it manually.
   2. Remove the buffer using the magnetic rack.
   3. Repeat the washing step.
   4. Add a volume of lysis buffer equal to the initial volume of slurry medium, mix by flicking the tube, and dispense the medium uniformly into as many 1.5 mL tubes as there are samples.
4. Bead blocking
   1. Remove the buffer using the magnetic rack and add 600 µL of a 0.25 mg/mL solution of yeast tRNA prepared in lysis buffer.
   2. Incubate for 1 h at room temperature on a rotating wheel.
   3. Remove the tRNA solution using the magnetic rack.
   4. Add 600 µL of lysis buffer and wash by mixing manually.
   5. Repeat the washing step and discard the buffer.

6. Bead loading

1. Prepare 200 µg of RNA oligonucleotide in 600 µL of lysis buffer for each tube containing the initial 100 µL slurry medium (now blocked beads).
2. Add the oligo to the beads and incubate for 1 h at room temperature while rotating.
3. Remove the solution, add 600 µL of lysis buffer, and wash the beads twice by rotating the tubes for 5 min at room temperature.
4. Discard the buffer.
   ​NOTE: Never vortex the beads, but flick instead. Limit the number of pipetting steps, unless necessary. When possible, use cut, 1 mL tips. The amount of bead slurry medium/sample depends on the binding capability of the beads and the starting amount of the total proteins. If the RNA oligo is predicted to have a significant amount of secondary structure, we recommend to first denature it at 80 °C for 10 min and then cool it down slowly at room temperature or refold it by incubating it at 30 °C for 1 h. It is suggested to recover the RNA oligo after bead loading and determine the concentration left, in order to optimize the amount required for loading and to evaluate the possibility of reusing the RNA.

7. Protein binding on beads

NOTE: From now on, when possible, perform the steps at 4 °C.

1. Take a 5% volume from the 600 µL protein solution and keep it as INPUT (IN) for further analysis (1.5 mg of proteins are dissolved in 600 µL, so 5% corresponds to 30 µL and 75 µg of proteins).
2. Add the remaining protein mix to each tube of loaded beads and leave slowly rotating overnight at 4 °C.

8. Washing of nonspecific binders

1. Remove the unbound fraction using the magnetic rack. Save 5% of the volume and label it as FLOWTHROUGH (FT) (the unbound volume is ca. 600 µL, so again keep 30 µL for further analysis).
2. Add 1 mL of wash buffer 1 to the beads and leave it rotating for 5 min at 4 °C.
3. Discard the buffer.
4. Repeat steps 8.2 and 8.3.
5. Add 1 mL of wash buffer 2 to the beads and leave it rotating for 5 min at 4 °C.
6. Discard the supernatant.

9. Elution of specific binders

1. Add 100 µL of elution buffer 1 or elution buffer 2 to the beads.
2. Mix manually by flicking and incubate for 5 min at room temperature.
3. Place the tubes in a thermomixer and shake vigorously for 5 min at 95 °C.
4. Place the tube into the magnetic rack and collect the eluted fraction into a clean tube.
5. Quickly spin the beads with a bench centrifuge to maximize the recovery of the eluate.
6. Save 5% of the total ELUATE (EL) volume for further analyses (total volume is 100 µL, so separate 5 µL into another tube).
7. If needed, the protein concentration can be determined as in section 4, by using elution buffer as a blank.
   NOTE: It is recommended to refrain from adding DTT to the elution buffer until protein concentration has been determined. If protein quantification is not required, or if reducing agent-compatible protein quantification kits are available, 1 mM DTT can be added to the elution buffer from the start. In this protocol, both elution buffer 1 (containing 1 M NaCl) and elution buffer 2 (with 2 M NaCl) were tested. No difference in the elution efficiency of the target protein was observed with increased ionic strength, but it is recommended to test both conditions before establishing the most appropriate buffer. If a high presence of salt in the elution buffer represents a limitation for further analysis, the very low amount of detergents in the elution buffer allows for buffer-exchange. As an alternative, the eluate can be diluted to reach the desired salt concentration.

10. Identification of protein binders by mass spectrometry

1. Acetone precipitation
   1. Concentrate the eluted proteins by diluting it fourfold in cold (-20 °C) acetone.
   2. Vortex and incubate the tube at -20 °C overnight.
   3. Spin at 17,000 x g for 30 min at 4 °C.
   4. Gently remove the supernatant and allow the acetone to evaporate until the pellet has completely dried.
2. In-solution protein digestion
   1. Dissolve the protein pellet by adding 50 µL of denaturation buffer.
   2. Add DTT to a final concentration of 5 mM, allowing protein reduction for 30 min at 55 °C.
   3. Cool down the samples at room temperature and proceed with the protein alkylation reaction, adding iodoacetamide (IAA) at a concentration of 10 mM for 15 min.
   4. Digest the proteins using a suitable enzyme (trypsin, LysC) and incubate the samples overnight at 37 °C.
   5. Stop the digestion by adding 1 µL of 10% trifluoroacetic acid (TFA).
   6. Clean up and concentrate the peptides on a custom reversed phase C18 micro-column, as previously described¹⁹.
   7. Elute peptides from the C18 tip with Buffer B.
   8. Remove the organic component using a vacuum centrifuge and resuspend the peptides in 5 µL of 0.1% formic acid for further analysis.
      NOTE: Alternatively, protein digestion can be performed "on-beads" immediately after washing nonspecific binders (steps 8.1-8.6), thus making the protocol faster. However, it is advisable to test the efficiency of the enzyme working "on-beads" immobilized-proteins compared to standard in solution digestion, in order to guarantee the optimal experimental protein sequence coverage.
3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
   1. Plug in the analytical column (C18-stationary phase) and keep it at 45 °C during the time of the run.
   2. Connect the column to the outlet of a six-port rotary valve of the LC pump through a capillary finger-tight fitting (20 µm x 550 mm) in a single-column configuration.
   3. Adjust the LC settings as follows:
      1. Load the peptides under controlled pressure (980 bar) in Buffer A.
      2. Apply a 5%-20% Buffer B gradient at 300 nL/min over 59 min, followed by a 20%-30% Buffer B gradient over 15 min and a 30%-65% Buffer B gradient over 5 min.
      3. Add a wash step by increasing the concentration of Buffer B up to 95% over 5 min plus a 5 min isocratic step at 95% Buffer B.
   4. Operate the mass spectrometer in data-dependent acquisition (DDA) mode to switch automatically between MS and MSMS events.
   5. Define a loop count equal to 15 using an automatic gain control (AGC) target value of 3 x 10⁶ and 1 x 10⁵ for the MS and MSMS events, respectively.
   6. Set the maximum allowed ion accumulation time to 20 ms for MS with a resolution of 60 K, and 100 ms for MSMS with a resolution of 15 K.
   7. Perform a high collision dissociation (HCD) fragmentation experiment using a normalized collision energy of 28%, with a dynamic exclusion time of 20 s.
   8. Operate the source parameters as follows:
      Spray voltage: 1.7 kV
      Capillary voltage: 275 °C
      Neither a sheath nor auxiliary gas used
      NOTE: In this protocol, ultra-high performance liquid chromatography (UHPLC) mass spectrometric (MS) analysis has been specifically performed using an LC single column setup, coupled to a hybrid triple quadrupole orbitrap instrument (Table of Materials). Other LCMS systems can be used, but an adaption of parameters is recommended.
4. Data analysis
   1. Use the Load button to import the raw files.
   2. Define the experiment names by clicking on the Set Experiment button.
   3. Enter the group-specific parameters section to specify all theparameters related to identification:
      Enzyme used for digestion: Trypsin/P
      Missed cleavages: up to three
      Fixed modification: Carbamidomethylation
      Variable modification: N-acetyl (Protein), Oxidation (M)
   4. Upload an updated FASTA file, available from public databases such as UniprotKB.
   5. Specify the correct parse rules according to the source of the chosen database.
   6. Define a parentage false discovery rate (FDR) value = 1 for both proteins and peptides.
   7. Add the label free quantification (LFQ) option in the Label Free Quantification tab.
   8. Keep the minimum LFQ ratio count at two.
      NOTE: Here, we describe data analysis using MaxQuant²⁴ and Perseus software²⁵ to perform protein quantification and subsequent statistical analysis, respectively. However, data analysis can be performed with any other commercially available or free bioinformatic. FDR is estimated using a target-decoy database-based approach²⁶. Peptide and protein FDR equal to 0.01 means that peptides and proteins identified are expected to contain 1% of false positives.
5. Statistical analysis
   1. Load the proteingroups.txt file to perform statistical analysis at the protein level.
   2. Define the LFQ values as main columns.
   3. Remove "reverse" and "contaminants" by filtering rows based on the categorical column.
   4. Use the categorical annotation rows to group the different experimental conditions.
   5. Reduce the data matrix, selecting the number of valid values in each of the previously defined groups.
   6. Select the statistical test that better suits the experimental conditions (i.e., t-test, multiple sample test ANOVA).
   7. Determine a cut-off for significant hints with an FDR-based calculation. Typically, both 0.01 and 0.05 are accepted as thresholds for the adjusted p value.
   8. Visualize the results of differential analyses based on t-test statistics using a volcano plot representation.
   9. Export the final matrix in .txt format for further editing of the final result table.
      NOTE: The configuration folder contains a FASTA file with proteins such as keratins that are considered common contaminants in global proteomic experiments, which are flagged with a + in the output table. In the present study, having two sample conditions, a t-test is used for the statistical analysis.

11. Results validation by western blot

1. Sample preparation
   1. Add the appropriate volume of 4x sample loading buffer to each aliquot of IN, FT, and EL.
   2. Boil the samples for 5 min at 95°C.
   3. Fast-spin to recover evaporated sample from the top of the tubes.
2. SDS-PAGE and gel transfer
   1. Load samples on a 4%-12% denaturing polyacrylamide gel.
   2. Run the gel with MES SDS running buffer for 1.5 h at 120 V.
   3. Transfer the gel on a nitrocellulose membrane with a semi-dry transfer cassette, following the manufacturer instructions. We recommend a 10 min transfer at 15 V.
3. Immunodetection
   1. Block the membrane with 10% bovine serum albumin (BSA) for 1 h at room temperature while gently agitating.
   2. Add the primary antibody prepared in 5% BSA in TBST, according to the manufacturer's instructions. Leave overnight at 4 °C or for 1 h at room temperature under gentle agitation.
   3. Wash the membrane three times with TBST, each time for 5 min.
   4. Add the secondary antibody prepared in TBST for 1 h at room temperature under agitation.
   5. Wash the membrane three times with TBST, each time for 5 min.
   6. Visualize the results using a blot imager.
      NOTE: For detecting TDP-43, a known binder of our RNA, recombinant rabbit monoclonal antibody was used and left with the membrane overnight at 4 °C. As a secondary antibody, the anti-rabbit IgG horseradish peroxidase (HRP) was used, but a fluorescent secondary antibody would work as well. To visualize the antibodies on the membrane, the membrane was incubated with the Clarity Western ECL substrate for 1 min, before imaging with the ChemiDoc imaging system.